biomolecules

Review
The COP9 Signalosome: A Multi-DUB Complex
Wolfgang Dubiel 1,2, * , Supattra Chaithongyot 1 , Dawadschargal Dubiel 1 and
Michael Naumann 1
1 Institute of Experimental Internal Medicine, Medical Faculty, Otto von Guericke University, Leipziger Str. 44,
39120 Magdeburg, Germany; supattra.chaithongyot@med.ovgu.de (S.C.); ddubiel@med.ovgu.de (D.D.);
naumann@med.ovgu.de (M.N.)
2 School of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative Drug Target Research,
Xiamen University, Xiang’an South Road, Xiamen 361102, China
* Correspondence: wolfgang.dubiel@med.ovgu.de




Received: 4 June 2020; Accepted: 13 July 2020; Published: 21 July 2020


Abstract: The COP9 signalosome (CSN) is a signaling platform controlling the cellular ubiquitylation
status. It determines the activity and remodeling of ~700 cullin-RING ubiquitin ligases (CRLs),
which control more than 20% of all ubiquitylation events in cells and thereby influence virtually any
cellular pathway. In addition, it is associated with deubiquitylating enzymes (DUBs) protecting CRLs
from autoubiquitylation and rescuing ubiquitylated proteins from degradation. The coordination of
ubiquitylation and deubiquitylation by the CSN is presumably important for fine-tuning the precise
formation of defined ubiquitin chains. Considering its intrinsic DUB activity specific for deneddylation
of CRLs and belonging to the JAMM family as well as its associated DUBs, the CSN represents a
multi-DUB complex. Two CSN-associated DUBs, the ubiquitin-specific protease 15 (USP15) and
USP48 are regulators in the NF-κB signaling pathway. USP15 protects CRL1β-TrCP responsible for IκBα
ubiquitylation, whereas USP48 stabilizes the nuclear pool of the NF-κB transcription factor RelA upon
TNF stimulation by counteracting CRL2SOCS1 . Moreover, the CSN controls the neddylation status of
cells by its intrinsic DUB activity and by destabilizing the associated deneddylation enzyme 1 (DEN1).
Thus, the CSN is a master regulator at the intersection between ubiquitylation and neddylation.

Keywords: COP9 signalosome; ubiquitylation; cullin-RING ubiquitin ligases; DUBs; USP15;

USP48; DEN1

1. Introduction
The COP9 signalosome (CSN) is a multiprotein complex representing a hallmark of eukaryotic cells.
The CSN was discovered as a repressor of constitutive photomorphogenesis (COP) in Arabidopsis [1,2]
and first isolated from cauliflower [3,4]. Purification from mammalian cells characterized the complex
as signaling particle (signalosome) possessing homology to the 26S proteasome lid [5–7]. In Mammalia,
core CSN is composed of six proteasome-COP9-initiation factor 3 (PCI) and two Mov34-and-Pad1p
N-terminal (MPN) domain subunits [8,9], essential for CSN function. The 3.8 Å resolution CSN
crystal structure based on human recombinant subunits provided detailed information about the
subunit-subunit interactions [10]. The PCI domain proteins oligomerize via their winged-helix
subdomains in the order of CSN7-CSN4-CSN2-CSN1-CSN3-CSN8, forming a horseshoe-like structure
(Figure 1). The MPN domain heterodimer (CSN5, CSN6) is situated on top of the helical bundle formed
by the C-terminal α-helices of each CSN subunit [10]. This architecture is shared by paralog complexes
of the CSN: the 26S proteasome lid and the translation initiation factor 3 (eIF3) [10,11].

Biomolecules 2020, 10, 1082; doi:10.3390/biom10071082 www.mdpi.com/journal/biomolecules

Biomolecules 2020, 10, x1082
FOR PEER REVIEW 22 of
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Figure 1. The COP9 signalosome (CSN) and its associated deubiquitylating enzymes (DUBs) and
Figure 1. The COP9 signalosome (CSN) and its associated deubiquitylating enzymes (DUBs) and
deneddylating enzyme 1 (DEN1). The structure of the CSN was obtained by cryo-electron microscopy
deneddylating enzyme 1 (DEN1). The structure of the CSN was obtained by cryo-electron microscopy
using native CSN purified from human red blood cells or from mouse B8 fibroblasts [12]. The localization
using native CSN purified from human red blood cells or from mouse B8 fibroblasts [12]. The
of CSN subunits, the “Helical Bundle” and the “Horseshoe” structure [10] is indicated. The crystal
localization of CSN subunits, the “Helical Bundle” and the “Horseshoe” structure [10] is indicated.
structure of USP15 is shown with its catalytic core (green) [13]. CSN5 is involved in CSN-USP15/Ubp12
The crystal structure of USP15 is shown with its catalytic core (green) [13]. CSN5 is involved in CSN-
interaction [14]. In addition, in A. nidulans USP15/UspA is presumably associated with the entire helical
USP15/Ubp12 interaction [14]. In addition, in A. nidulans USP15/UspA is presumably associated with
bundle [15]. So far, there is no crystal or cryo-structure of USP48 available. The crystal structure of
the entire helical bundle [15]. So far, there is no crystal or cryo-structure of USP48 available. The
DEN1/NEDP1 is from Shen et al. [16]. In human cells DEN1 mostly interacts with the N-terminus of
crystal structure of DEN1/NEDP1 is from Shen et al. [16]. In human cells DEN1 mostly interacts with
CSN1, whereas in fungi it preferentially binds to CSN7 [17].
the N-terminus of CSN1, whereas in fungi it preferentially binds to CSN7 [17].
The CSN is substantially more heterogeneous than indicated by the structure of the eight-core
The CSN
subunits. It isisassociated
substantially more
with heterogeneous
kinases and modified than byindicated by the structure
phosphorylation [18–20].of the
Foreight-core
instance,
subunits. It is associated with kinases and modified by phosphorylation
in response to DNA damage causing double strand breaks the ATM kinase phosphorylates CSN3 [21] [18–20]. For instance, in
response to DNA damage causing double strand breaks the ATM kinase
and UV damage leads to modification of CSN1 [22] with consequences for DNA damage repair. phosphorylates CSN3 [21]
and
Thus,UV damage leads to modification of CSN1
phosphorylation/dephosphorylation in [22] with consequences
response for DNA damage
to signaling processes produces repair. Thus,
a dynamic
phosphorylation/dephosphorylation
heterogeneity of CSN complexes. Moreover, in response to recently
it has signalingbeen processes
recognizedproduces
that a afraction
dynamic of
heterogeneity
cellular CSN contains a non-essential, non-canonical component called CSNAP [23–25]. A cellular
of CSN complexes. Moreover, it has recently been recognized that a fraction of further
CSN containssource
unexplored a non-essential, non-canonical
of heterogeneity component
is provided called
by the fact CSNAP
that several [23–25].
CSN core A further
subunits unexplored
occur as
source of heterogeneity
paralogs/isoforms [26,27]. isCSNprovided
subunit by the fact
isoforms that several
originate from gene CSN core subunits
duplication [26] or occur
from use as
paralogs/isoforms [26,27]. CSN subunit isoforms originate from gene duplication
of alternative translation start sites as shown for CSN8A and CSN8B [28]. They are integrated into [26] or from use of
alternative
distinct CSNtranslation start coexist
variants, which sites asinshown
cells. Infor CSN8A and
Arabidopsis, CSN8B
the CSN [28]. They
variants CSNCSN5Aare integrated
and CSNCSN5B into
distinct CSN variants, which coexist in cells. In Arabidopsis,
confer different physiological functions [2] and in human cells CSN the CSN variants
CSN7A CSN
and CSN
CSN5A and CSNCSN5B
CSN7B have distinct
confer
roles indifferent
adipogenic physiological functions
differentiation [2] and in human cells CSNCSN7A and CSNCSN7B have distinct
[26,29].
roles in adipogenic differentiation [26,29].
2. The CSN Belongs to the Deubiquitylating Enzymes (DUBs)
2. The CSN Belongs to the Deubiquitylating Enzymes (DUBs)
The ~100 DUBs encoded by the human genome belong to the following families: the ubiquitin
The ~100protease
(Ub)-specific DUBs encoded by the humanhydrolase
(USP), Ub-C-terminal genome belong
(UCH),to the following families:
JAB1/MPN+/MOV34 the ubiquitin
protease (JAMM),
(Ub)-specific protease (USP), Ub-C-terminal hydrolase (UCH),
ovarian tumor protease (OTU), Josephin [30], the novel motif interacting with Ub-containing JAB1/MPN+/MOV34 protease
DUB
(JAMM),
(MINDY) [31]ovarian tumor
and the protease
recently (OTU),
discovered Josephin [30],
ZUFSP/ZUP1 family the novel
[32]. DUBs motif interacting
are pivotal withofUb-
regulators the
containing DUB (MINDY) [31] and the recently discovered ZUFSP/ZUP1
Ub system involved in protein turnover, signaling, sorting and trafficking [33]. Whereas most DUBs family [32]. DUBs are
pivotal regulators
are cysteine of the
proteases, theUb system
CSN involved
and its paralogincomplex
protein lidturnover, signaling, sorting
are metalloproteases of theandJAMMtrafficking
family
[33]. Whereas most
with conserved DUBs
His, Asp andare
Ser cysteine
coordinating proteases, the Zn
a catalytic CSN [30].
2+ and CSN5its paralog complex
is the only lid are
CSN subunit
metalloproteases
possessing the JAMM of the JAMM
motif. Of family with
note, free conserved
CSN5 His,[12,34]
is inactive Asp and Ser coordinating
similar to its paralog asubunit
catalytic Zn2+
RPN11
[30]. CSN5 is the only CSN subunit possessing the JAMM motif. Of note, free CSN5 is inactive [12,34]
similar to its paralog subunit RPN11 of the 26S proteasome lid [35]. Furthermore, within the CSN
Biomolecules 2020, 10, 1082 3 of 11

of the 26S proteasome lid [35]. Furthermore, within the CSN complex, CSN5 is in an auto-inhibited
state, the Ins-1 conformation [10]. Active CSN5 specifically removes NEDD8 from isopeptide-bonds
with conserved Lys residues of cullins. The deneddylating activity of the CSN is activated by its
substrates, neddylated cullin-RING-Ub ligases (NEDD8-CRLs) [36], which change the conformation
of the active site called induced fit [37]. Structural data provide evidence for coordinated domain
changes of CSN2, CSN4, and CSN7 and the CSN5-CSN6 heterodimer induced by neddylated CRL4A
converting CSN5 into its active conformation [37]. Obviously, the CSN complex is an essential platform
for CSN5 to act as a DUB. Purified mammalian CSN is a DUB specific for NEDD8 and unable to cleave
Ub-AMC [12]. Using polyubiquitylated CUL4A as substrate and mutated CSN5, deubiquitylating
activity was detected in crude Flag-CSN pulldowns from HeLa cells demonstrating the existence of
CSN associated DUBs. Since wildtype CSN5 exhibited different deubiquitylating activity as compared
to mutant CSN5, it was assumed that CSN5 has a deubiquitylating activity on its own [38]. However,
the data might just reflect an impact of CSN5 on associated DUBs.
Neddylation [39] and deneddylation constitute a regulatory cycle, in which deneddylation
inactivates CRLs [37,40] and NEDD8 conjugation stimulates CRL activity by multiple mechanisms [41–44].
Furthermore, CSN-mediated deneddylation is a prerequisite for the exchange of hundreds of substrate
receptors (SRs) including F-box and BTB-domain SRs [45–47] as part of a rapid adaptation to altered
protein degradation requirements [48]. In this process the CSN cooperates with Cullin-Associated and
Neddylation-Dissociated 1 (CAND1) to accelerate the exchange of SRs to optimize the CRL network in
response to fluctuations in substrate availability [45,46,49–54].
Recently, a potent and specific inhibitor of CSN-mediated deneddylating activity has been
discovered, which is called CSN5i-3 [55]. The compound blocks cullin deneddylation and traps CRLs
in the neddylated state. CSN5i-3 affects the viability of many tumor cells and suppresses growth of
human xenografts in mice [55]. This excellent tool stimulates current and future research on CSN
mechanisms and tumor therapy.
In summary, the CSN is a DUB of the JAMM family, controlling the Ub-dependent protein
degradation mediated by CRLs, which is essential for maintaining processes such as cell cycle [56],
DNA repair [57] and differentiation [48].

3. The CSN and Its Paralog 26S Proteasome Lid Cooperate with Diverse DUBs
Analyses of the CSN isolated from different cells by chromatography [14], pulldowns [29],
immunoprecipitation [58] as well as density gradient centrifugation [17] revealed its association
with additional DUBs, such as USP15 and USP48 and presumably other DUBs as well as with
DEN1/NEDP1/SENP8, a member of the SENP family (Figure 1). Thus, the CSN occurs as a multi-DUB
complex. USP15 and USP48 belong to the USPs, the largest DUB family, with more than 50 members
in Mammalia [30]. Both are characterized by one (USP48) or two (USP15) UBL domains and are
involved in multiple unrelated biochemical pathways and cellular responses. USP15 activity has
been associated with parkin-mediated mitochondrial ubiquitylation and mitophagy [59] and the
nuclear factor erythroid 2-related factor 2 pathway in an anti-oxidant response [60]. USP15 has also
been shown to stabilize the CRL component RBX1 [61] as well as adenomatous polyposis coli (APC),
a subunit of the β-catenin destruction complex [62] and to regulate transforming growth factor-β
signaling [63]. There are less reports on USP48 engagements. USP48 stabilizes TRAF2 influencing the
E-cadherin-mediated adherens junctions [64]. Whether all these USP15 and USP48 activities need CSN
association is not clear at the moment. In the review we focus on CSN associated USP15 and USP48
and their functions in the NF-κB pathway as well as on DEN1, an associated deneddylase.
RPN11, the paralog to CSN5, is the intrinsic DUB of the lid, which also belongs to the JAMM-DUB
family. Similar to CSN5, the Ins-1 loop of RPN11 undergoes conformational transition from inactive to
active state, which is, in case of RPN11, directed by Ub and ATP [65]. In analogy to the CSN5-CSN6
heterodimer, RPN11 partners with another MPN domain protein, RPN8, possessing an inactive JAMM
domain. The activated lid specifically cleaves Ub chains and promotes protein degradation by the
Biomolecules 2020, 10, 1082 4 of 11

26S proteasome. A deneddylating activity of the lid was not reported. In the 19S regulatory particle
the lid cooperates with USP14, a DUB of the USP family, and UCH37 of the UCH family. USP14 and
UCH37 are not integral subunits of the 26S proteasome, they assist the lid in removing ubiquitin from
substrates to ensure the function of the proteasome [66]. Interestingly, USP14 and UCH37 bind to
RPN1 and to RPN13, respectively, which are, in addition to the RPN10, substrate receptors of the 19S
regulatory particle [67], which provide a versatile binding platform for various ubiquitin chains [68].
Thus, a coordinated deubiquitylation of incoming substrates within the 19S regulator is presumably
necessary for proper function of the 26S proteasome, which is accomplished by the cooperation of lid,
USP14 and UCH37. However, since the lid is not directly associated with USP14 and UCH37, it is just
part of a multi-DUB complex within the 19S particle.
Unfortunately, so far just few data are published on a possible intrinsic DUB activity of the other
paralog complex, the eIF3 [69] and nothing is known about associated DUBs.
Similar structural principles as in the CSN and the lid are mirrored in the BRCA1-A complex in
which the active JAMM domain DUB, BRCC36, interacts with the inactive JAMM protein ABRAXAS,
whereas in the BRISC complex BRCC36 is supported by ABRO1 [70]. The main substrates of both
complexes are Lys63-chains. Their functions, however, are completely different. Whereas BRCA1-A
complex serves in DNA double-strand break repair sequestering BRCA1, the BRISC complex is
involved in immune signaling. Thus, in this case complexes confer different targeting and specific
regulatory functions to BRCC36 DUB, although the substrate remains the same [70]. In case of CSN
and lid, the context of their multi-protein complexes provides substrate specificity to the DUBs as well
as specific functions.

4. CSN-DUB Interactions and Their Role in NF-κB Regulation

The CSN is a signaling platform and in cooperation with the associated USP15 (Ubp12p in
Schizosaccharomyces pombe or UspA in Aspergillus nidulans) it is involved in the NF-κB pathway.
Binding of USP15 to the CSN seems to be conserved. According to studies in S. pombe the CSN is
necessary for efficient transport of Ubp12p to the nucleus. In absence of CSN5, Ubp12p is entirely
lost from the CSN [14]. In A. nidulans, UspA presumably interacts with the helical bundle of the CSN
(see Figure 1) [15]. In human cells, the association between the CSN and USP15 was demonstrated by
diverse methods [61]. However, to date, the exact subunits and the CSN variants involved in USP15
interaction are not known.
The CSN recruits USP15 to protect components of CRLs during the remodeling process of the
E3 ligase complexes [47,61]. Recently, UspA-dependent protection of the F-box protein F-box23 was
shown in A. nidulans [15]. Interestingly, F-box23 is presumably the paralog of β-TrCP in mammalian
cells, the SR of CRL1β-TrCP responsible for the ubiquitylation of IκBα [71]. By stabilizing the F-box23,
the CSN-associated UspA reduces protein levels of the fungal NF-κB-like velvet domain protein VeA,
which coordinates differentiation and secondary metabolism [15]. Further, USP15 deubiquitylates
IκBα enhancing its stability. Thus, USP15 plays a critical role in the re-accumulation of IκBα in the
cytoplasm after induction of the NF-κB pathway, which contributes to the termination of the NF-κB
signal [72,73] (Figure 2). CSN1 has a specific role in this context: the N-terminus of CSN1 contacts the
CRL1β-TrCP , whereas its C-terminus directly interacts with IκBα [74]. Studies on atherogenesis also
highlight the CSN function in NF-κB regulation demonstrating the protection of IκBα from degradation
by endothelial CSN5/CSN leading to reduction of NF-κB activation upon tumor necrosis factor (TNF)
stimulation [75].
CK2 activity including localization, turnover and association with other proteins [79]. Most
interestingly, the CSN is associated with CK2 [18], highlighting its role as a signaling platform
presumably controlling the activity of associated DUBs. In this context, USP15 also is phosphorylated
by CSN-associated
Biomolecules CK2 [61]. However, the function of this modification is still obscure.
2020, 10, 1082 5 of 11

Figure 2. The COP9 signalosome (CSN) partners with ubiquitin-specific protease 15 (USP15) and
Figure 2. TheinCOP9
48 (USP48) signalosome
the NF-κB (CSN)
signaling partners
pathway. (1)with
Uponubiquitin-specific
stimulation, theprotease
activated15 IκB
(USP15)
kinaseand 48
(IKK)
(USP48) in the NF-κB signaling pathway. (1) Upon stimulation, the activated
complex phosphorylates IκBα, which is subsequently recognized by β-TrCP, the substrate receptor of IκB kinase (IKK)
complex
the CRL1. phosphorylates IκBα, which
(2) The CRL1β-TrCP is subsequently
ubiquitylates (K48) IκBα, recognized by β-TrCP,
which becomes the substrate
consequently receptor
degraded of
by the
the
26SCRL1. (2) The (3)
proteasome. CRL1 β-TrCP
Due to the ubiquitylates
degradation(K48)
of IκBα, IκBα, which
NF-κB becomesand
is released consequently
translocateddegraded by theto
to the nucleus
26S proteasome.
activate (3) Due
NF-κB target to the
genes degradation
including IκBα.of(4)IκBα, NF-κB
De novo is released
synthesized andcan
IκBα translocated to the nucleus
be phosphorylated again,
to(5)activate NF-κB target genes including
but the CSN regulates the activity of CRL1 IκBα. (4)
β-TrCPDe novo synthesized IκBα can be phosphorylated
by deneddylation and (6) CSN-associated USP15,
again,
which(5) but the stabilization
promotes CSN regulates the
and activity of CRL1
re-accumulation
β-TrCP by deneddylation and (6) CSN-associated
of IκBα, thereby (7) terminating NF-κB activation.
USP15,
In a latewhich
responsepromotes stabilization
at the chromatin, andCRL2
(8) the SOCS1 /ECSSOCS1
re-accumulation of targets
IκBα, thereby (7) RelA
the nuclear terminating NF-κB
for degradation.
activation.
However, In a late response attranscription
NF-κB-dependent the chromatin, (8) thecould
activity CRL2be SOCS1/ ECSSOCS1 targets the nuclear RelA for
sustained by the CSN-associated USP48
degradation. However,
activity. (9) Herein, USP48NF-κB-dependent transcription
deubiquitylates RelA, activity could
which stabilizes RelA atbethesustained
chromatin. by the CSN-
associated USP48 activity. (9) Herein, USP48 deubiquitylates RelA, which stabilizes RelA at the
The ubiquitin-specific protease 48 (USP48) was identified as a predominately nuclear
chromatin.
CSN-associated DUB by co-immunoprecipitation [58]. It stabilizes the nuclear pool of the NF-κB
Collectively,
transcription USP15
factor RelAandupon USP48 are the onlybytwo
TNF stimulation CSN-associated
counteracting DUBs
the ECS /CRL2SOCS1 E3soligase
SOCS1characterized far.
Additional work2)is[58,76].
activity (Figure necessary
Theto understand
catalytic domain their exact
of the interplay with
deubiquitylase distinct
USP48 CSNthevariants.
directs In
interaction
addition, unexplored
with the Rel homologyDUBsdomain interacting
of RelA [77].with specific CSNCSN-associated
Mechanistically, variants are challenging
USP48 trims for future
K48-linked
investigations.
ubiquitin chainsFor this purpose,
of RelA, additional
thus controlling CSN associated
the NF-κB activity. DUBs already identified in mass
spectrometry experiments
Its trimming activity [80] have to be
is enhanced by verified by appropriate
CK2-mediated methods and
phosphorylation suchinactivated
as chemical by
crosslinking mass spectrometry
dephosphorylation [58]. CK2 or is aBio-ID,
highlyfollowed
conservedby binding
protein studies and mutational
kinase possessing analysis.
brought substrate
specificity with pro-proliferative and anti-apoptotic activities. It is constitutively active and
5.messenger-independent
CSN-DEN1: The Interplay [78]. of TwoisMajor
There Deneddylases
a multitude of mechanisms potentially contributing to regulate
CK2 activity including localization, turnover and association with other proteins [79]. Most interestingly,
the CSN is associated with CK2 [18], highlighting its role as a signaling platform presumably controlling
the activity of associated DUBs. In this context, USP15 also is phosphorylated by CSN-associated
CK2 [61]. However, the function of this modification is still obscure.
Biomolecules 2020, 10, 1082 6 of 11

Collectively, USP15 and USP48 are the only two CSN-associated DUBs characterized so far.
Additional work is necessary to understand their exact interplay with distinct CSN variants.
In addition, unexplored DUBs interacting with specific CSN variants are challenging for future
investigations. For this purpose, additional CSN associated DUBs already identified in mass
spectrometry experiments [80] have to be verified by appropriate methods such as chemical crosslinking
mass spectrometry or Bio-ID, followed by binding studies and mutational analysis.

5. CSN-DEN1: The Interplay of Two Major Deneddylases

The physical interaction between the CSN and DEN1 is conserved from fungi to human [17].
In A. nidulans, direct protein-protein interaction was determined predominately between DEN1 and
CSNG/CSN7, but, in addition, with CSNA/CSN1, CSNE/CSN5 and CSNF/CSN6. In human cells,
DEN1 preferentially binds to the N-terminus of CSN1 but there is a weak interaction with CSN2 as
well (Figure 1) [17]. To date, it is unknown whether DEN1 specifically binds to a certain CSN variant.
Moreover, the exact function of the CSN-DEN1 interaction is still a matter of debate.
DEN1 is a cysteine protease belonging to the Ub-like protease (ULP) family closely related to
DUBs [16,30,81,82]. DEN1 crystal structure (Figure 1) reveals the classical catalytic triad, Cys, His and
Asp, and the structural basis for its high specificity for NEDD8 [16]. DEN1 can process C-termini
of NEDD8 and deconjugates hyperneddylated cullin 1 (CUL1) [83]. However, in fungi NEDD8,
processing seems not be an important function of DEN1. Processed NEDD8 was not sufficient to
rescue a denA deletion mutant [17]. Moreover, the efficiency of DEN1 to cleave NEDD8-CUL1 substrate
in vitro is about 100 times lower as compared to the CSN [12]. Therefore, DEN1 is not just another
deconjugase of neddylated cullins but also most likely deneddylates additional proteins modified by
NEDD8. In A. thaliana, DEN1 removes NEDD8 from a subunit of the NEDD8 activating enzyme (NAE).
It is assumed that DEN1 regulates the cellular level of free NEDD8, an important control factor for the
neddylation/deneddylation cycle [84]. In A. nidulans, DEN1/DENA is necessary for appropriate light
reaction. In contrast to the CSN, which is responsible for the fungal sexual development, DEN1 is
needed for asexual development. Deletion of denA revealed a role for the deneddylase in asexual spore
formation during limited pyrimidine supply [17].
In human cells, knockout of DEN1 [85] as well as oxidative stress [86] cause accumulation
of NEDD8 conjugates, although neddylation of CRLs might even show a significant decrease [87].
Interestingly, human microvascular endothelial cells lacking DEN1 were unable to neddylate CUL1 and
subsequently were not able to activate NF-κB or HIF-1α with consequences for vascular inflammatory
response. These studies provide evidence for a role of DEN1 in fine-tuning of the inflammatory
response [88].
Recently it was found that under conditions of inhibited DEN1, poly-NEDD8 chains are formed [89].
Under normal conditions, NEDD8 chains are degraded by DEN1. If DEN1 is suppressed as under
oxidative stress conditions or by knockout, a specific NEDD8 trimer accumulates, which interacts with
poly(ADP-ribose) polymerase 1 (PARP1) and attenuates PARP1 activation. Since hyper-activation
of PARP1 results in cell death, accumulation of the tri-NEDD8 prevents this cell fate [89]. Therefore,
DEN1 has a pivotal role in the regulation of cell death. Whether this DEN1 activity is dependent on its
interaction with the CSN is not clear.
One important function of CSN-DEN1 interaction is the initiation of DEN1 degradation [17].
Interestingly, the stability of DEN1 depends on its phosphorylation status. As shown in fungi, DEN1
degradation is regulated by interaction with the CSN as well as by the interplay of phosphorylation
and the phosphatase DipA [90]. Thereby, the CSN balances cellular deneddylase activity, which is
essential for fungal development and most likely for prevention of human diseases.

6. Concluding Remarks
Recently it became clear that the coupling of Ub conjugating and deconjugating machineries is a
common principle contributing to the regulation of complex signaling networks [91]. Ub ligases and
Biomolecules 2020, 10, 1082 7 of 11

DUBs regulate the status of protein ubiquitylation, which is crucial for essential cellular processes such
as cell cycle progression, DNA repair, signal transduction, protein quality control and differentiation [30].
The CSN is a signaling platform equipped with kinases that coordinates the action of CRLs and DUBs.
The member of the JAMM family reduces ubiquitylation of CRLs via deneddylation, thereby protecting
CRL components from autoubiquitylation. To optimize this function, the CSN cooperates with USP15
to stabilize CRLs for reassembly and adaptation to changing cellular requests. Further, CSN-associated
USP15 directs deubiquitylation and stabilization of CRL-substrate IκBα, whereas CSN-associated USP48
stabilizes the nuclear pool of RelA, thereby facilitating timely induction and shutoff of NF-κB target
genes. In addition, the CSN controls deneddylation activity in cells by determining DEN1 stability.

Author Contributions: Conceptualization, W.D. and M.N.; Writing—Original Draft, W.D., M.N., S.C. and D.D.;
Writing—Review & Editing, W.D., M.N., S.C. and D.D.; Visualization, W.D., D.D., M.N., S.C., Funding Acquisition,
W.D. and M.N. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded in part by grant 31770813 of the Natural Science Foundation of China to W.D,
by grant ZS/2016/04/78155 of the European Union program ERDF (European Regional Development Fund) and
Ministry of Economy, Science and Digitalization in Saxony Anhalt within the Center of Dynamic Systems to W.D.
and M.N., and by grant 361210922/RTG 2408 of the German Research Foundation to M.N.
Acknowledgments: The work was in part supported by grant 31770813 of the Natural Science Foundation of
China to W.D, by grant ZS/2016/04/78155 of the European Union program ERDF (European Regional Development
Fund) and Ministry of Economy, Science and Digitalization in Saxony Anhalt within the Center of Dynamic
Systems to W.D. and M.N., and by grant 361210922/RTG 2408 of the German Research Foundation to M.N.
Conflicts of Interest: The authors declare no conflicts of interest.

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